Nonpolio enterovirus, a member of the Picornaviridae family, is composed of VP1-4 structural proteins and a positive single-stranded RNA of about 7,400 bases as a genome, and the entire genome is translated as a single polypeptide from the 5′ end thereof, which is cleaved by virus-encoded proteases into a set of individual proteins (see FIG. 1).
The enterovirus is a major causative agent which is responsible for a wide spectrum of human diseases ranging from mild aseptic meningitis to life-threatening dilated cardiomyopathy (see [Grist, N. R. et al., Prog Med Virol, 24:114-157, 1978; Muir, P., Br J Biomed Sci, 50:258-271, 1993; and Rotbart, H. A., Clin Infect Dis, 20:971-981, 1995]), but no effective preventive or therapeutic treatments against this virus infection are currently available.
RNA interference (RNAi) is a natural ‘gene expression knock-down’ process, which occurs in a sequence-specific manner (see [Dykxhoorn, D. M. et al., Nat Rev Mol Cell Biol, 4:457-467, 2005; and Fire, A. et al., Nature, 391:806-811, 1998]). This event involves a small interfering RNA (siRNA) of approximately 19-23 nt which specifically triggers catalytic degradation of complementary mRNAs via RNA-induced silencing complex (RISC) (see [Elbashir, S. M. et al., Nature, 411:494-498, 2001; Fire, A. et al.; and Zamore, P. D. et al., Cell, 101:25-33, 2000]).
There have accumulated numerous evidences showing that both chemically synthesized siRNAs and vectors expressing short hairpin RNAs (shRNA) can also induce RNAi in vitro and in vivo (see [Bernstein, E. et al., Rna, 7:1509-1521, 2001; Dykxhoom, D. M. et al.; and Hasuwa, H. et al., FEBS Lett, 532:227-230, 2002]). Moreover, recent studies have suggested that RNAi can be used as a promising novel platform technology for the discovery of effective antiviral drugs (see [Dave, R. S. et al., Rev Med Virol, 13:373-385, 2003; and Stevenson, M., Nat Rev Immunol, 3:851-858, 2003]). These studies have consistently demonstrated that siRNAs are capable of effectively inhibiting the replication of a variety of viruses, such as hepatitis virus, poliovirus, or influenza virus under diverse experimental conditions. It was further demonstrated that siRNAs exhibit dramatic antiviral effects against coxsackievirus B3 (CVB3) in permissive cells (see [Ahn, J. et al., Intervirology, 46:245-251, 2003; Merl, S. et al., Circulation, 111:1583-1592, 2005; Schubert, S. et al., J Mol Biol, 346:457-465, 2005; and Yuan, J. et al., J Virol, 79:2151-2159, 2005]).
Yuan et al. examined the protective abilities of five CVB-specific siRNAs against CVB3 infection in both HeLa cells and murine cardiomyocytes, and found that the siRNA which targets viral protease 2A is the most effective (see Yuan, J. et al.).
Nonpolio enteroviruses include coxsackieviruses (CV) and echoviruses (Echo), which are CVA serotype (CVA1 to CVA3, CVA5 to CVA24), CVB serotype (CVB1 to CVB6), and 33 Echo serotypes (see [van Regenmortel, M. H. V. et al., Virus taxonomy. Academic Press, 2000]). In other words, nonpolio enteroviruses are characterized by: a number of distinct serotypes along with high genetic variability; high mutation rate during replication due to its lack of proof-reading capability; and ineffective siRNA manifestation when point mutations occur within the target region. Therefore, viral genome's variability and instability should be carefully considered in order to successfully utilize RNAi as antiviral therapeutics (see [Gitlin, L. et al., Nature, 418:430-434, 2002; and Gitlin, L. et al., J Virol, 79:1027-1035, 2005]).
Accordingly, the present inventors have endeavored to develop a multi-enteroviral targeting siRNA, which has a significant antiviral activity against a variety of nonpolio enteroviruses.